A variety of post-translational modifications including methylation, sulfation, phosphorylation, lipid addition and glycosylation are performed on proteins expressed by higher eukaryotes. Glycosylation involves the covalent attachment of sugar moieties to specific amino acids and is one of the most common and important posttranslational modification for recombinant proteins. Protein glycosylation plays a role in multiple functions, including protein folding and quality control, molecular trafficking and sorting, and cell surface receptor interaction. Many of the secreted proteins, membrane proteins and proteins targeted to vesicles or certain intracellular organelles are known to be glycosylated.
While glycosylation can take many forms, N-linked glycosylation is the most common. N-linked glycosylation involves addition of oligosaccharides to an asparagine residue found in certain recognition sequences in proteins (e.g., Asn-X-Ser/Thr). N-linked glycoproteins contain standard branched structures which are composed of mannose, galactose, N-acetylglucosamine and neuramic acids. N-linked glycosylation of the Fc domain of recombinantly expressed therapeutic antibodies is a critical posttranslational modification. Typical therapeutic antibodies have complex glycoforms possessing fucosylated bi-antennary glycans with a trimannosyl core capped by an N-acetylgalactosamine (GlcNAc), galactose, and N-acetylneuraminic acid (Neu5Ac) residue on each branch. Other glycoforms may be afucosylated, galactosylated, sialylated, have terminal or bisecting GlcNAc, have high mannose (5-9 residues), etc.
Glycosylation can affect therapeutic efficacy of recombinant protein drugs. It is well known that variations in Fc glycosylation can affect Fc-mediated effector functions. Some glycoforms, such as galactosylation and sialylation, are desirable for decreasing immunogenicity, and others, such as afucosylation, bisecting GlcNAc residues, and high mannose glycans, enhance antibody-dependent cellular cytotoxicity (ADCC) activity.
Glycosylation is important in the determination of the structure and function of therapeutic antibodies. It determines binding capabilities and often determines the recognition and processing of the antibody once it is introduced in a therapeutic application. In the case of galactosylation and fucosylation, they determine the complement dependent cytotoxicity (CDC) activity and ADCC functions, respectively, that they influence.
The level of β-galactosylation is related to more “mature” glycoforms. Galactose addition is one of the last stages of glycosylation that takes place in the Golgi apparatus before secretion. Terminal galactose is needed for sialylation, which may be the final step in the glycosylation of some proteins. Galactose also serves as a ligand for galactose binding proteins and is the basis of a variety of antigenic responses which are related to carbohydrate content. Galactose has also been shown to impact the conformation of the protein in solution. (Furukawa and Sato, (1999) Biochimica et Biophysica Acta (BBA), 1473 (1), pages 54-86 and Houde et al., (2010) Molecular and Cellular Proteomics, 9(8), pages 1716-1728.
Fucosylation also takes place in the Golgi apparatus as part of the maturation of the protein prior to secretion. If a protein is fucosylated it typically happens before galactosylation in the glycosylation pathway. However, fucosylation is not necessary for galactosylation to proceed (Hossler et al., (2009) Glycobiology, 19(9), pages 936-949).
The influence of glycosylation on bioactivity, pharmacokinetics, immunogenicity, solubility and in vivo clearance of therapeutic glycoproteins have made monitoring and control of glycosylation a critical parameter for biopharmaceutical manufacturing. Therefore, methods for manipulating the level of glycan content of therapeutic proteins would be beneficial.
There is a need in the pharmaceutical industry to manipulate and control the level of glycan content of recombinant therapeutic glycoproteins and methods for accomplishing such without significant impact on cell growth, viability and productivity would be useful. The invention provides a method for manipulating the fucosylated glycan content on a recombinant protein by regulating copper and manganese content and pH in cell culture medium.